1. Field of the Invention
The present invention relates to centrifugation and more particularly to the control of rotor speed for controlling concentration variation in a centrifuge chamber.
2. Description of Related Art
Essentially, centrifugation is a process for separating particles suspended in a solution. In biological applications, the particles are usually macromolecules, cells, DNA fragments, etc. There are two primary types of centrifuge procedures, one being "preparative" which is to isolate specific particles, and the other being "analytical" which involves measuring the physical properties of a sedimenting particle.
The device used for centrifugation is a centrifuge which includes a rotor that supports several containers or centrifuge tubes, of sample solution for rotation about a common spin axis. As the rotor spins in the centrifuge, centrifugal force is applied to each particle in the sample solution; each particle will sediment at a rate which is proportional to the centrifugal force applied. The viscosity of the sample solution and the physical properties of the particle also affect the sedimentation rate of each individual particle. For a given centrifugal force, density and liquid viscosity, the sedimentation rate of the particle is proportional to its size (molecular weight), and to the difference between its density and the density of the solution.
One of many methods of centrifugal separation is differential centrifugation, or pelleting. In this method, the centrifuge tube is filled initially with a uniform mixture of sample solution. Through centrifugation, one obtains a separation of two fractions including a pellet containing the sedimented material, and supernatant solution of the unsedimented material. The pellet is a mixture of all of the sedimented components.
Another method of separation is by density gradient centrifugation, a method somewhat more complicated than differential centrifugation, but one which has compensating advantages. Not only does the density gradient method permit the complete separation of several or all of the components in a mixture according to their densities but, it also permits analytical measurements to be made. The density gradient method involves a supporting column of "density gradient" fluid whose density increases toward the bottom of the tube. The density gradient fluid consists of a suitable low molecular weight solute in a solvent in which the sample particles can be suspended.
There are two widely used methods of density gradient centrifugation: rate zonal and isopycnic. In the rate zonal technique, a sample solution containing particles to be separated is layered on a preformed gradient column. Under centrifugal force, the particles will begin sedimenting through the gradient toward the bottom of the centrifuge tube and separate into zones along the tube, each zone consisting of particles characterized by their sedimentation rate. The zones continue to move down the tube with time. To achieve a rate zonal separation, the density of the sample particles must be greater than the density at any specific position along the gradient column, in order for the particles to be able to continue to move down the tube. The run must be terminated before any of the separated zones reaches the bottom of the tube. Otherwise, two or more separated zones will mix at the bottom of the tube.
In the isopycnic technique, the density gradient column encompasses the whole range of densities of the sample particles. Each particle will sediment or float toward a position in the centrifuge tube at which the gradient density is equal to its own density, and there it will remain in equilibrium. The isopycnic technique, therefore, separates particles into zones on the basis of their density differences.
Density gradients can be formed manually by layering density gradient material of gradually decreasing densities in the centrifuge tube. In the isopycnic procedure, it is sometimes easier to start with a uniform solution of the sample and the gradient material. Under the influence of centrifugal force, the gradient material redistributes in the tube so as to form the required concentration (and density) gradient. This is often referred to as the selfgenerating gradient technique in which a continuous density gradient is formed when the diffusion of the gradient material towards the rotor spin axis balances the sedimentation away from the spin axis at each radial location along the centrifuge tube. Meanwhile, sample particles, which are initially distributed throughout the tube, sediment or float to their isopycnic positions. This self-generating gradient technique often requires long hours of centrifugation.
Generally, centrifugal separation can be effected in less time by centrifuging at higher rotor angular velocity. However, it is limited by the condition that the centrifugal force of the mass of the rotor and its contents at high rotor velocity not exceed the yield stress of the rotor. Furthermore, the rotor angular velocity is also limited by the condition that the solution of the gradient material not attain saturation at the outermost location of the centrifuge tube at any time during centrifugation. This is to avoid the possibility of salt crystallization or precipitation, which involves a process of accumulation of mass as the salt transforms from the dissolved to solid crystalline phase. This can lead to possible rotor failure due to excessive stress on the rotor by the dense crystalline salt.
Rotor manufacturers typically provide manuals that contain information regarding the top speed at which the rotor can be safely run for an indefinite period of time, for a given loading concentration, without reaching the gradient salt precipitation threshold. Referring to FIG. 1, W.sub.p represents the speed at which precipitation will never occur for an indefinite period of time for a particular loading density. In the past, centrifugation run has been carried out at a single speed W.sub.p to safely operate within the precipitation threshold (line 12). A more efficient method would be to run the rotor at the highest speed W.sub.y within the yield stress limit of the rotor, until precipitation is expected to occur at time T.sub.c at which time the rotor speed is reduced to the value W.sub.p which would then not allow precipitation to occur for an indefinite length of time (line 14). Using this method, the total elapsed centrifugation time required for a particular state of separation is less than that in the case of the single speed run. One can also compare the efficiency of the two methods by comparing the integrals of W.sup. 2 under the operating lines 12 and 14, the larger integral for the same elapsed time being representative of a more efficient centrifugation.
Recently, a new technique was proposed by Chulay et al in U.S. Pat. No. 4,941,868 which utilizes a dynamic simulation of gradient salt sedimentation to predict the elapsed time at which the precipitation threshold is reached for a number of discrete speeds. The technique requires several speed reductions in coarse steps (line 16) during a run to maintain the gradient salt density within the precipitation threshold. The reduced speeds are designated by the manufacturer and are selected via trial and error to decrease run time. This technique significantly increases the average speed of the rotor above that found in both of the previously described techniques. The amount of time required to attain separation is therefore substantially decreased.
While the technique described in the Chulay et al patent has been found to be efficient, the process for finding curve 16 is not automated; furthermore the rotor speed has not been optimized to produce the shortest run time. In recent days when centrifugation has quite a few competing technologies for macromolecular separation, automated minimization of the amount of time required for centrifugal separations has become commercially important. Because the technique described in the Chulay et al. patent requires a trial and error approach to selecting the speeds at which centrifugation is to be run, the highest possible speed at a particular elapsed time very often has not been selected.